Week 8 - enzyme interactions Flashcards
Define what an enzyme is
- Specific protein catalysts that increase the rate of chemical reactions without being consumed by the reaction
How may enzymes be targets for therapeutic drugs?
- drugs could act as an alternative substrate for the enzyme competing with the specific substrate for binding to the active site and in general inhibiting its activity
- Alternatively, a drug could act by inhibiting the enzymes activity through binding to other sites on the enzyme in allosteric modulation
Besides the inhibition and modulation of an enzyme, how else may drugs interact with an enzyme?
- Some drugs must undergo modification to convert thm from the inactive (prodrug) form to an active form
What is the key physical characteristic of drugs?
What impact does this have on the body?
- Drugs, by design, are lipophillic which ensures that they can be readily absorbed from the GI tract and taken up into the circulation
- However, the lipophillicity means that they would tend to be continually reabsorbed in the kidney tubules where the tissue is more oil-rich than the urine
- This would result in the accumulation of toxins in cell membranes causing damage or prolonged pharmacological activity
- How this is avoided is discussed in card #5
How are the issues arising from a drugs lipophillic nature avoided?
- Lipophillic substances undergo biotransformation to make them mor ehighly water soluble
- Some drugs, at physiological pH 7.4, exist in their fully ionised form and can be secreted without any modifications
- Most drugs undergo sequential metabolic changes categorised as phase I and phase II reactions
Briefly describe which group of enzymes is responsible for phase I metabolism of drugs
How must this be considered in drug discovery?
- CYP450 group are the most abundant enzymes in the liver and are responsible for the metabolism (phase I) of 70% of drugs in the body
- Dominant CYP in the liver is CYP3A4, which is 70% of all the CYP450 enzymes in the liver, which has a broad substrate specificity meaning it metabolises a vast range of molecules
- Screening of the CYP450 enzymes is important in early drug discovery
Define “drug metabolism” or “xenobiotic metabolism”
- Drug / xenobiotic metabolism is the chemical modification of substances, including drugs or xenobiotics such as toxic chemicals, through the action of a specific family of enzymes
- They convert lipophillic/hydrophobic substances that can acumulate in lipid membranes into more hydrophillic metabolites which can be excreted from the body
Define “xenobiotic”
How are xenobiotics metabolised?
Why is metabolism required?
- A xenobiotic is a chemical compound, such as drug, pesticide, carcinogen, that is foreign to a living organism and enters from the external environment
- They are metabolised by pathways of biotransformation that can be found in most organisms, including bacteria which suggests that these pathways have ancient origin
- These reactions are required to detoxify poisonous compounds and remove them from the body before they can cause harm
What does the rate of drug metabolism have on a drug?
- Rate at which the conversion from a lipophillic/hydrophobic substance to a more hydrophillic substance impacts the duration and intensity of a drug’s pharmacological activity
How many phases does drug metabolism take place in?
Describe these phases
- Drug metabolism takes place in three phases
- Phase I involves enzymes, such as cytochrome P450 oxidases, which make xenobiotics more hydrophillic and reactive by introducing polar groups into their structures
- Once modified, nucleophile metabolites are conjugated to polar compounds through glucuronidation, acetylation or sulphonation and electrophile metabolites are modified by glutathione conjugation in phase II reactions which are catalysed by transferase enzymes such as glutathione S-transferases
- In phase III reactions, efflux transporters recognise the conjugated metabolites and use the hydrolysis of ATP to pump them out of the cell against the concentration gradient
Outline phase I metabolism of xenobiotics
- Phase I involves enzymes, such as cytochrome P450 oxidases, which make xenobiotics more hydrophillic and reactive by introducing polar groups into their structures
Outline phase II metabolism of xenobiotics
Give an example of the enzyme involved
- Once modified, nucleophile metabolites are conjugated to polar compounds through glucuronidation, acetylation or sulphonation and electrophile metabolites are modified by glutathione conjugation in phase II reactions
- Catalysed by transferase enzymes such as glutathione S-transferases
Outline phase III metabolism of xenobiotics
- In phase III reactions, efflux transporters recognise the conjugated metabolites and use the hydrolysis of ATP to pump them out of the cell against the concentration gradient
Give an overview of the cytochrome P450 enzymes, what they catalysed and what they are responsible for
- Cytochrome P450 (CYP450) superfamily are a group of enzymes that catalyse the oxidation of lipid soluble xenobiotics
- Role if CYP450 enzymes is to catalyse the metabolism of endogenous substances such as lipids and steroidal hormones, as well as exogenous xenobiotics including drugs and toxic chemicals
Outline a typical reaction catalysed by CYP450 enzymes
- Typical reaction catalysed by CYP450 enzymes is a monooxygenase reaction
- This involves the binding of substrate and molecular oxygen to CYP and the tranfer of one oxygen atom into a substrate (RH) while the second oxygen atom is reduced to water
- RH + O2 + NADPH + H+ → ROH + H2O + NADP+
Outline the structure and location of the CYP450 enzymes
- CYP450’s are membrane bound haemoproteins that are located in the smooth endoplasmic reticulum
- In the liver they are often closely associated with enzymes wich catalyse phase II conjugation reactions, e.g. UDP-glucuronyl tranferase (UDPGT)
How many CYP enzymes are there in humans?
How are they classified?
- There are a large number of CYPs in nature and 57 CYPs in humans, of which 15/57 are responsible for metabolising drugs and other xenobiotics
- Classified on their amino acid homology and can be divided into families
- 18 families have been identified in humans (CYP1-18)
- There are subfamilies based on sequence homology and are termed CYP1A, CYP2A/B/C/etc.
How is substrate specificity of CYP isoforms determined?
- Specificity of the CYP isoforms is determined by the amino acid sequences of their binding sites
- A change of one amino acid can be crucial to the specificity of the molecules that these enzymes metabolise
What are the similarities shared by all CYP enzymes?
State the first similarity
- There are seven similarities that all CYPs share
1. Exist in a lipid-rich environment so tend to be located in the lipophillic membrane of the smooth ER, positioned within membranes so their access channels can be easiliy accessed by lipophillic substrates
What are the similarities shared by all CYP enzymes?
State the second similarity
- CYPs have highly conserved rigid regions within their active sites where iron containing haem groups reside
- these are surrounded by more flexible binding regions
What are the similarities shared by all CYP enzymes?
State the third similarity
- The binding area is variable and so determines the specificity of the substrate
What are the similarities shared by all CYP enzymes?
State the fourth similarity
- The ability of the iron within the active site to gain or lose electons is the reason that CYPs can catalyse oxidation and reduction reactions